Regulation of lung development by FGF9 signaling pathways Summary Lung disease is the fourth leading cause of death and disability in the United States. The origins of lung disease often begin during development or early in life and developmental mechanisms are often redeployed in acquired disease. Thus, it is essential to unravel the complex mechanisms that regulate lung development to understand the pathogenesis of developmental, genetic, and acquired lung disease. Moreover, to design therapeutic approaches for repair or regeneration, and to treat cancers and other disorders, it is necessary to determine how signaling networks coordinate development and become dysregulated in disease. We have identified Fibroblast Growth Factor 9 (FGF9) as a developmental signaling molecule that is expressed in lung mesothelium and epithelium that has an essential role in regulating lung mesenchyme and a proposed secondary role in regulating lung epithelium during embryonic development. In lung mesenchyme, we identified a feed-forward regulatory network that links mesenchymal FGF9-FGFR1/2 signaling and Wnt/?- catenin signaling. We also showed that deregulated expression of FGF9 disrupts lung development through expansion of mesenchymal and epithelial compartments, as in the case of Pleuropulmonary Blastoma (PPB), a heritable pediatric lung cancer that arises from lung mesenchyme. During the previous funding period we found that in PPB, which is initiated by loss of function mutations in DICER1, that lung epithelial micro RNAs function to suppress Fgf9 expression during development. Loss of these regulatory micro RNAs, through mutation in DICER1 and resultant deregulated expression of FGF9 contributes to the mesenchymal hyperplasia and possibly the cystic lesions that are characteristic of Type I PPB. We also found that deregulated expression of FGF9 in the adult results in an FGF receptor (FGFR) 3- dependent rapid induction of epithelial proliferation with progression to adenocarcinoma. These studies highlight the necessity to tightly regulate FGF9 expression and activity in the embryo and in the adult. Here, we propose experiments that will uncover mechanisms that regulate Fgf9 and that will identify how FGF9 and Wnt/?-catenin signaling regulates epithelial and mesenchymal target tissues. Because micro RNA control of Fgf9 expression appears to primarily function during very early (pseudoglandular) stages of lung organogenesis, we hypothesize that additional mechanisms are necessary to control (suppress) Fgf9 at later stages. In Aim 1 we will examine the regulation of Fgf9 expression by the histone methyltransferase, EZH2, a component of the Polycomb Repressive Complex 2 (PRC2), as a mechanism to place long lasting repressive marks on the Fgf9 gene. Aims 2 and 3 will examine signaling mechanisms that are downstream of FGF9 in mesenchyme and epithelium, respectively. This is important because the identity of the downstream target genes and the transcriptional mechanisms that couple FGF9 and Wnt/?-catenin signaling to the regulation of these target genes are not known.